Fig. 1

Effects of MLN4924 on ependymal Foxj1 transcription factor expression. a Representative IHC staining of control (Ctrl) and MLN4924-treated primary EC cultures, labeled with acetylated tubulin (A-tub) antibody and DAPI. Scale bar: 50 µm. b Quantification of multiciliated cell numbers as a fraction of DAPI⁺ nuclei. *P < 0.008, Wilcoxon two-sample test, n = 5, z = 1.107. c Representative IHC staining from Ctrl and MLN4924-treated primary EC cultures, labeled with Foxj1 antibody and DAPI. Scale bar: 30 µm. d Quantification of number of Foxj1⁺ cells as a fraction of DAPI nuclei in untreated and MLN4924-treated primary EC cultures. *P < 0.008, Wilcoxon two-sample test, n = 5, z = 1.331. e IHC staining of differentiated primary EC cultures without (Ctrl) or with MLN4924 treatment for 12 h, labeled with Foxj1 antibody and DAPI. Note the increase in relative fluorescence intensity for Foxj1 in MLN4924-treated cultures. Scale bar: 20 µm. f Western blot analysis showing relative Foxj1 protein levels from primary EC cultures without (Ctrl) or with MLN4924 treatment for 12 h. Actin is used as protein loading control. g Quantification of relative amounts of Foxj1 protein from western blot analyses in f. *P < 0.03, Wilcoxon two-sample test, n = 4, z = 1.316. h IHC staining of fixed ependymal wholemounts from P28 animal without (Ctrl) or with MLN4924 treatment for 6 h, labeled with Foxj1 and DAPI. Scale bar: 10 µm. i Quantification of cell numbers with indicated Foxj1 IHC fluorescence intensities, presented as histogram comparing untreated (blue) vs. 24 h MLN4924 treatment (orange). Dashed lines represent Gaussian fit distribution curve. *P < 0.0001, unpaired Student’s t test, n = 172 in each group, t₁₇₁ = 49.780. Box plots show mean (+), median (−), quartiles (boxes), range (whiskers)